Biochemistry and Pharmacology - Research Publications
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ItemIntegrated Transcriptomic and Metabolomic Mapping Reveals the Mechanism of Action of Ceftazidime/Avibactam against Pan-Drug-Resistant Klebsiella pneumoniae(AMER CHEMICAL SOC, 2023-10-25)Here, we employed an integrated metabolomics and transcriptomics approach to investigate the molecular mechanism(s) of action of ceftazidime/avibactam against a pan-drug-resistant K. pneumoniae clinical isolate from a patient with urinary tract infection. Ceftazidime/avibactam induced time-dependent perturbations in the metabolome and transcriptome of the bacterium, mainly at 6 h, with minimal effects at 1 and 3 h. Metabolomics analysis revealed a notable reduction in essential lipids involved in outer membrane glycerolipid biogenesis. This disruption effect extended to peptidoglycan and lipopolysaccharide biosynthetic pathways, including lipid A and O-antigen assembly. Importantly, ceftazidime/avibactam not only affected the final steps of peptidoglycan biosynthesis in the periplasm, a common mechanism of ceftazidime action, but also influenced the synthesis of lipid-linked intermediates and early stages of cytoplasmic peptidoglycan synthesis. Furthermore, ceftazidime/avibactam substantially inhibited central carbon metabolism (e.g., the pentose phosphate pathway and tricarboxylic acid cycle). Consistently, the dysregulation of genes governing these metabolic pathways aligned with the metabolomics findings. Certain metabolomics and transcriptomics signatures associated with ceftazidime resistance were also perturbed. Consistent with the primary target of antibiotic activity, biochemical assays also confirmed the direct impact of ceftazidime/avibactam on peptidoglycan production. This study explored the intricate interactions of ceftazidime and avibactam within bacterial cells, including their impact on cell envelope biogenesis and central carbon metabolism. Our findings revealed the complexities of how ceftazidime/avibactam operates, such as hindering peptidoglycan formation in different cellular compartments. In summary, this study confirms the existing hypotheses about the antibacterial and resistance mechanisms of ceftazidime/avibactam while uncovering novel insights, including its impact on lipopolysaccharide formation.
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ItemNo Preview AvailableModel-informed dose optimisation of polymyxin-rifampicin combination therapy against multidrug-resistant Acinetobacter baumannii.(Elsevier BV, 2023-09)OBJECTIVES: Antimicrobial resistance is a major global threat. Because of the stagnant antibiotic pipeline, synergistic antibiotic combination therapy has been proposed to treat rapidly emerging multidrug-resistant (MDR) pathogens. We investigated antimicrobial synergy of polymyxin/rifampicin combination against MDR Acinetobacter baumannii. METHODS: In vitro static time-kill studies were performed over 48 h at an initial inoculum of ∼107 CFU/mL against three polymyxin-susceptible but MDR A. baumannii isolates. Membrane integrity was examined at 1 and 4 h post-treatment to elucidate the mechanism of synergy. Finally, a semi-mechanistic PK/PD model was developed to simultaneously describe the time course of bacterial killing and prevention of regrowth by mono- and combination therapies. RESULTS: Polymyxin B and rifampicin alone produced initial killing against MDR A. baumannii but were associated with extensive regrowth. Notably, the combination showed synergistic killing across all three A. baumannii isolates with bacterial loads below the limit of quantification for up to 48 h. Membrane integrity assays confirmed the role of polymyxin-driven outer membrane remodelling in the observed synergy. Subsequently, the mechanism of synergy was incorporated into a PK/PD model to describe the enhanced uptake of rifampicin due to polymyxin-induced membrane permeabilisation. Simulations with clinically utilised dosing regimens confirmed the therapeutic potential of this combination, particularly in the prevention of bacterial regrowth. Finally, results from a neutropenic mouse thigh infection model confirmed the in vivo synergistic killing of the combination against A. baumannii AB5075. CONCLUSION: Our results showed that polymyxin B combined with rifampicin is a promising option to treat bloodstream and tissue infection caused by MDR A. baumannii and warrants clinical evaluations.
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ItemUntargeted metabolomics to evaluate polymyxin B toxicodynamics following direct intracerebroventricular administration into the rat brain(ELSEVIER, 2022)There is a dearth of studies focused on understanding pharmacokinetics, pharmacodynamics and toxicodynamics of polymyxins following direct administration to the central nervous system (CNS). In this study, for the first time, untargeted metabolomics were employed to ascertain the perturbations of brain metabolism in the rat cerebral cortex following direct brain injection of 0.75 mg/kg polymyxin B (1 and 4 h) through the right lateral ventricle. In the right cortex metabolome, ICV polymyxin B induced a greater perturbation at 1 h compared to negligible effect at 4 h. Pathway enrichment analysis showed that sphingolipid, arginine, and histidine metabolism, together with aminoacyl-tRNA biosynthesis were significantly affected in the right cortex metabolome. Furthermore, intracerebroventricular (ICV) polymyxin B dysregulated the two arms (CDP-choline and CDP-ethanolamine) of the Kennedy pathway that governs the de novo biosynthesis of neuronal phospholipids. Importantly, the key intermediates of metabolic pathways that maintain cellular redox balance (e.g., glutathione metabolism) and mitochondrial function (e.g., electron transport chain) were markedly depleted. The abundance of key metabolites (e.g., N-acetyl-l-glutamate) associated with diverse CNS disorders (e.g., neurodegenerative disease) were also significantly perturbed. The biological significance of these metabolic perturbations on the CNS includes impaired oxidant-antioxidant balance, impaired neuronal lipid homeostasis and mitochondrial dysfunction. Furthermore, ICV polymyxin B caused a significant alteration in the abundance of several metabolic biomarkers associated with cerebral ischemia, oxidative stress as well as certain neurological disorders. These findings may facilitate the development of new pharmacokinetic/pharmacodynamic strategies to attenuate polymyxins ICV related CNS toxicities and stimulate the discovery of safer next-generation polymyxin-like lipopeptide antibiotics.
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ItemDrug Repurposing Approaches towards Defeating Multidrug-Resistant Gram-Negative Pathogens: Novel Polymyxin/Non-Antibiotic Combinations(MDPI, 2022-12)Multidrug-resistant (MDR) Gram-negative pathogens remain an unmet public health threat. In recent times, increased rates of resistance have been reported not only to commonly used antibiotics, but also to the last-resort antibiotics, such as polymyxins. More worryingly, despite the current trends in resistance, there is a lack of new antibiotics in the drug-discovery pipeline. Hence, it is imperative that new strategies are developed to preserve the clinical efficacy of the current antibiotics, particularly the last-line agents. Combining conventional antibiotics such as polymyxins with non-antibiotics (or adjuvants), has emerged as a novel and effective strategy against otherwise untreatable MDR pathogens. This review explores the available literature detailing the latest polymyxin/non-antibiotic combinations, their mechanisms of action, and potential avenues to advance their clinical application.
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ItemIntegrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxin-rifampicin combination against Pseudomonas aeruginosa(BMC, 2022-10-30)BACKGROUND: Understanding the mechanism of antimicrobial action is critical for improving antibiotic therapy. For the first time, we integrated correlative metabolomics and transcriptomics of Pseudomonas aeruginosa to elucidate the mechanism of synergistic killing of polymyxin-rifampicin combination. METHODS: Liquid chromatography-mass spectrometry and RNA-seq analyses were conducted to identify the significant changes in the metabolome and transcriptome of P. aeruginosa PAO1 after exposure to polymyxin B (1 mg/L) and rifampicin (2 mg/L) alone, or in combination over 24 h. A genome-scale metabolic network was employed for integrative analysis. RESULTS: In the first 4-h treatment, polymyxin B monotherapy induced significant lipid perturbations, predominantly to fatty acids and glycerophospholipids, indicating a substantial disorganization of the bacterial outer membrane. Expression of ParRS, a two-component regulatory system involved in polymyxin resistance, was increased by polymyxin B alone. Rifampicin alone caused marginal metabolic perturbations but significantly affected gene expression at 24 h. The combination decreased the gene expression of quorum sensing regulated virulence factors at 1 h (e.g. key genes involved in phenazine biosynthesis, secretion system and biofilm formation); and increased the expression of peptidoglycan biosynthesis genes at 4 h. Notably, the combination caused substantial accumulation of nucleotides and amino acids that last at least 4 h, indicating that bacterial cells were in a state of metabolic arrest. CONCLUSION: This study underscores the substantial potential of integrative systems pharmacology to determine mechanisms of synergistic bacterial killing by antibiotic combinations, which will help optimize their use in patients.
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ItemInwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity(SPRINGER BASEL AG, 2022-06)Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.
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ItemNo Preview AvailableIn vitro evaluation of drug delivery behavior for inhalable amorphous nanoparticle formulations in a human lung epithelial cell model(ELSEVIER, 2021-03-01)Respiratory tract infections caused by multidrug-resistant (MDR) Gram-negative bacteria such as Pseudomonas aeruginosa are serious burdens to public health, especially in cystic fibrosis patients. The combination of colistin, a cationic polypeptide antibiotic, and ivacaftor, a cystic fibrosis transmembrane regulator (CFTR) protein modulator, displays a synergistic antibacterial effect against P. aeruginosa. The primary aim of the present study is to investigate the transport, accumulation and toxicity of a novel nanoparticle formulation containing colistin and ivacaftor in lung epithelial Calu-3 cells. The cell viability results demonstrated that ivacaftor alone or in combination with colistin in the physical mixture showed significant toxicity at an ivacaftor concentration of 10 μg/mL or higher. However, the cellular toxicity was significantly reduced in the nanoparticle formulation. Ivacaftor transport into the cells reached a plateau rapidly as compared to colistin. Colistin transport across the Calu-3 cell monolayer was less than ivacaftor. A substantial amount (46-83%) of ivacaftor, independent of dose, was accumulated in the cell monolayer following transport from the apical into the basal chamber, whereas the intracellular accumulation of colistin was relatively low (2-15%). The nanoparticle formulation significantly reduced the toxicity of colistin and ivacaftor to Calu-3 cells by reducing the accumulation of both drugs in the cell and potential protective effects by bovine serum albumin (BSA), which could be a promising safer option for the treatment of respiratory infections caused by MDR P. aeruginosa.
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ItemNo Preview AvailableComparative metabolomics reveals key pathways associated with the synergistic activity of polymyxin B and rifampicin combination against multidrug-resistant Acinetobacter baumannii(PERGAMON-ELSEVIER SCIENCE LTD, 2021-02)Multidrug-resistant (MDR) Acinetobacter baumannii presents a critical challenge to human health worldwide and polymyxins are increasingly used as a last-line therapy. Due to the rapid emergence of resistance during polymyxin monotherapy, synergistic combinations (e.g. with rifampicin) are recommended to treat A. baumannii infections. However, most combination therapies are empirical, owing to a dearth of understanding on the mechanism of synergistic antibacterial killing. In the present study, we employed metabolomics to investigate the synergy mechanism of polymyxin B-rifampicin against A. baumannii AB5075, an MDR clinical isolate. The metabolomes of A. baumannii AB5075 were compared at 1 and 4 h following treatments with polymyxin B alone (0.75 mg/L, i.e. 3 × MIC), rifampicin alone (1 mg/L, i.e. 0.25 × MIC) and their combination. Polymyxin B monotherapy significantly perturbed glycerophospholipid and fatty acid metabolism at 1 h, reflecting its activity on bacterial outer membrane. Rifampicin monotherapy significantly perturbed glycerophospholipid, nucleotide and amino acid metabolism, which are related to the inhibition of RNA synthesis. The combination treatment significantly perturbed the metabolism of nucleotides, amino acids, fatty acids and glycerophospholipids at 1 and 4 h. Notably, the intermediate metabolite pools from pentose phosphate pathway were exclusively enhanced by the combination, while most metabolites from the nucleotide and amino acid biosynthesis pathways were significantly decreased. Overall, the synergistic activity of the combination was initially driven by polymyxin B which impacted pathways associated with outer membrane biogenesis; and subsequent effects were mainly attributed to rifampicin via the inhibition of RNA synthesis. This study is the first to reveal the synergistic killing mechanism of polymyxin-rifampicin combination against polymyxin-susceptible MDR A. baumannii at the network level. Our findings provide new mechanistic insights for optimizing this synergistic combination in patients.
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ItemMechanisms Underlying Synergistic Killing of Polymyxin B in Combination with Cannabidiol against Acinetobacter baumannii: A Metabolomic Study(MDPI, 2022-04)Polymyxins have resurged as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As reports of polymyxin resistance in A. baumannii with monotherapy have become increasingly common, combination therapy is usually the only remaining treatment option. A novel and effective strategy is to combine polymyxins with non-antibiotic drugs. This study aimed to investigate, using untargeted metabolomics, the mechanisms of antibacterial killing synergy of the combination of polymyxin B with a synthetic cannabidiol against A. baumannii ATCC 19606. The antibacterial synergy of the combination against a panel of Gram-negative pathogens (Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) was also explored using checkerboard and static time-kill assays. The polymyxin B-cannabidiol combination showed synergistic antibacterial activity in checkerboard and static time-kill assays against both polymyxin-susceptible and polymyxin-resistant isolates. The metabolomics study at 1 h demonstrated that polymyxin B monotherapy and the combination (to the greatest extent) significantly perturbed the complex interrelated metabolic pathways involved in the bacterial cell envelope biogenesis (amino sugar and nucleotide sugar metabolism, peptidoglycan, and lipopolysaccharide (LPS) biosynthesis), nucleotides (purine and pyrimidine metabolism) and peptide metabolism; notably, these pathways are key regulators of bacterial DNA and RNA biosynthesis. Intriguingly, the combination caused a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids) compared to very minimal changes induced by monotherapies. At 4 h, polymyxin B-cannabidiol induced more pronounced effects on the abovementioned pathways compared to the minimal impact of monotherapies. This metabolomics study for the first time showed that in disorganization of the bacterial envelope formation, the DNA and RNA biosynthetic pathways were the most likely molecular mechanisms for the synergy of the combination. The study suggests the possibility of cannabidiol repositioning, in combination with polymyxins, for treatment of MDR polymyxin-resistant Gram-negative infections.
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ItemAn Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates(FRONTIERS MEDIA SA, 2022-03-15)Peptide-Peptide Nucleic Acid (PNA) conjugates targeting essential bacterial genes have shown significant potential in developing novel antisense antimicrobials. The majority of efforts in this area are focused on identifying different PNA targets and the selection of peptides to deliver the peptide-PNA conjugates to Gram-negative bacteria. Notably, the selection of a linkage strategy to form peptide-PNA conjugate plays an important role in the effective delivery of PNAs. Recently, a unique Cysteine- 2-Cyanoisonicotinamide (Cys-CINA) click chemistry has been employed for the synthesis of cyclic peptides. Considering the high selectivity of this chemistry, we investigated the efficiency of Cys-CINA conjugation to synthesize novel antimicrobial peptide-PNA conjugates. The PNA targeting acyl carrier protein gene (acpP), when conjugated to the membrane-active antimicrobial peptides (polymyxin), showed improvement in antimicrobial activity against multidrug-resistant Gram-negative Acinetobacter baumannii. Thus, indicating that the Cys-CINA conjugation is an effective strategy to link the antisense oligonucleotides with antimicrobial peptides. Therefore, the Cys-CINA conjugation opens an exciting prospect for antimicrobial drug development.